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Sporulation: Tetrad Dissection
Overview This protocol describes dissection of yeast tetrads. In our lab, we primarily use tetrad dissection for constructing strains for genetic and biochemical experiments. An accomplished yeast biologist can dissect a plate of spores in 20 minutes; for a beginner, 2 hours is not unusual. Tetrad dissection is a learnable skill. Your initial attempts will likely be frustrating. If you persevere, you will be rewarded by your new ability to wield one of the most powerful tools in the yeast geneticist's toolbox. When you first learn to do tetrad dissection, make sure to ask for help from someone in the lab who is experienced at doing it! It really helps to have someone with a practiced eye point out what a well-digested culture looks like, what a tetrad like under the dissecting scope, ect. Materials * β-glucuronidase (Sigma G7770, Stored in 4°C refrigerator. Comes as an aqueous solution in ~1.0 M ammonium sulfate with 3 mM sodium azide as preservative.) * Sporulated yeast culture * Sterile Water * "Dry" YPD dissection plates ** Everyone has a particular way that they like their plates for tetrad dissection. You are basically aiming for dry and level. To make dissection plates, add 25 ml YPD with a plastic strippette to plates on a very level surface. Once solid, invert. Let dry at room temperature for ~3 days. Bag. These plates are best after aging for a while. Protocol Digestion The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme β-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely. # Spin down 1 mL of sporulated culture at 10k rpm for 2 minutes. Dispose of the supernatant. # Resuspend in 200 μL sterile water. # Mix 17 μL washed culture with 3 μL β-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion. # Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time.) # Gently add 200 μL water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix. # Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads). # Mark the plate at opposite edges to indicate the center of the plate (see following diagram). # Resuspend the digested cells from your chosen timepoint. by gently tapping or pipetting (they will probably have settled while you were looking at the digestions under the microscope). Drip 20 μl down the imaginary center line you just indicated, beginning from one edge. # Let the digested culture fully absorb into the plate. # Dissect (see dissection section). If you dissect on a different day, you must begin with a fresh plate and set up a new dissection. The sporulation cultures are good for weeks, though the spore viability may start to decrease. Appearance of Tetrads Before digestion, tetrads are held tightly in a tetrahedron and it is often difficult ot see all four spores at once. After digestion they relax into a diamond shape. How to distinguish a tetrad from two budded cells adjacent to each other? # Generally, the spores of a tetrad are smaller than a budded cell. # Tetrads, if moved gently, will remain intact while two adjacent budded cells will not. # The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.